The Effect Of Lactacystin On The Inflammatory Factors IL-1? And TNF-a In Parkinson's Disease Model Cells Induced By Lactacystin

Purpose:Living by observing coumarin of Lactacystin induced neuronal PC12 cell activity and inflammatory cytokines IL-1?,influence of TNF-?,live with coumarin on neuronal PC12cell protection mechanism,to live with coumarin treating Parkinson's disease to provide experimental basis and theoretical basis.1.Material and method:1.Drug-contained serum was prepared:normal group(20),twin-80 group(10),celecoxib group(10),and only live coumarin group(10).Group of rats in normal saline lavage,polysorbate 80 group to 0.08%twain,80,2 ml/time,living by celecoxib and coumarin group rats to drug dosage according to the man and rats to the dose conversion formula to calculate,Mr Selebi yesterday cloth group carried out in accordance with the specification of adult dosage of every day routine conversion,live with coumarin group rats actual dosage according to the adult living by every 10g yinpian undertake convert the amount of 3 times[8].Administration time is 8:00~10:00,1 time per day,2ml/time/only,7days continuously.Finally,the blood was taken from the abdominal aorta after 2h,and the serum was isolated after 10min after centrifugation for 2h and 2000r/min.The 56?,30 min inactivated,0.22 mu m filter membrane filtration sterilization,placed-20?refrigerator.Set aside.2.Cell differentiation and culture of neurons:Rat adrenal chromaffin cells(PC12)after nerve growth factor(NGF)induced into neuronal PC12 cell,and then select the cell culture medium containing 15%horse serum,the logarithmic phase,according to the density of 1x 10~5/ml,vaccination in 96-well plates,including 100?l,to cultivate after 24 hours of adherent cell growth,Lactacystin and drug-containing serum concentration of screening.3.Determination of Lactacystin action concentration:Will Lactacystin mixture tendency for 1/l mother liquor,then Lactacystin mother liquid dilution of 0?mol/l,5?mol/l,10?mol/l and 20?mol/l final concentration added to the cell cultures,duration of 24h,determined by MTT tests each cell activity,and finally determine the sample Lactacystin induced neuronal PC12 cells become the best in the cell model of Parkinson's disease concentration of 5?mol/l.4.Screening of serum concentrations and time:The logarithmic phase of neuronal PC12 cell,according to the density of 1x 10~5/ml,vaccination in 96-well plates,including 100?l,to cultivate after 24 hours of adherent cell growth,the cell was divided into 16 group,blank control group,respectively(horse serum)and normal control group,the model of serum group,twain serogroup,celecoxib group,live with coumarin serum group,then the drug-containing serum groups other than the blank control group is divided into three concentration 10%,15%,20%group,replace the corresponding concentration of culture medium,protect time after 24h,48h,respectively,In addition to the blank control group and the normal serum control group,the other groups were added to the 5?mol/l Lactacystin for 24h.Finally,MTT was used to detect the relative survival rate of the cells,and the optimum serum concentration of 15%was determined,and the optimal time was24h.5.Grouping and administration:the experiment was divided into five groups,namely,normal control group,model group,twain group,selexib group,and coumarin group.With the density of 1x10~5/ml to part of the exponential phase of PC12 cells inoculated in two 96-well plates,part of vaccination in 6 orifice plate culture at the same time,the concentration of 15%selected different drug-containing serum culture after 24h,in addition to the normal control group groups respectively to join 5?mol/l Lactacystin,24h after collection of cells and the supernatant.6.Indicator detection and statistical processing:Cell activity was detected by MTT.The protein content of IL-1?and TNF-?was detected by ELISA.mRNA expression of IL-1?and TNF-?was detected by RT-PCR.SPSS19.0 statistical software was used to analyze the experimental results,and all the experimental data were used to indicate that the experimental data of multiple groups were analyzed with one-way anova,and p<0.05 was considered as statistically significant difference.Results:1.Determination of Lactacystin action concentration:The results showed that the absorbance value determined by MTT assay decreased gradually with the increase of Lactacystin concentration,that is,Lactacystin had significant dose-dependent inhibitory effect on cell activity.When Lactacystin had a concentration of 5?mol/l,the cell had the highest relative survival rate.Therefore,the selection concentration was 5?mol/l,and the effect of 24h was to induce the stimulation conditions of the neuron-like PC12 cells.2.Determination of the time and concentration of drug containing serum:It was selected to determine 5?mol/l Lactacystin effect 24h as the stimulus condition.With different concentrations of medicated serum intervention for cell protection,compared with the different concentrations of model group,the experimental results show that 15%medicated serum protection 24h,48h,cells OD value and relative survival rate increased significantly(P<0.01),while 15%concentration of serum medicated protect cells in the 24h OD value and relative survival rate higher than 48h.In line with the principle of the minimum serum concentration with the highest relative survival rate,the optimal condition was to choose a15%drug serum for 24h.3.The effect of serum on the activity of Lactacystin-induced PC12 cells:The results showed that there was no statistical difference between the cell OD value between the model group and the tween 80 group(p>0.05).The OD value of the model group and tween group was significantly lower than that in the control group(p<0.01).The OD value was higher than that in the model group(p<0.01),and the group(p<0.01)and the cecoxib group(p<0.01),but lower than the normal control group(p<0.01).The cell OD value of the cycloxib group was higher than that of the model group(p<0.01)and the group 80(p<0.01),but was lower than the normal control group(p<0.01)and the coumarin group(p<0.01).4.The effects of serum on the expression of IL-1?,TNF-?protein and IL-1?and TNF-?mRNA in the serum of Lactacystin induced neuron-like PC12 cells4.1 the effect of the expression of IL-1?,TNF-?protein and the expression of IL-1?and TNF-?in the cells of Lactacystin induced neuron-like PC12 cellsThe results showed that there was no statistical difference between the model group and the tween 80 group(p>0.05).Compared with the normal control group,the expression of IL-1?,TNF-?protein and IL-1?and TNF-?mRNA in the cells of the model group were significantly increased(p<0.01).4.2 effects of cecoxib on the expression of IL-1?,TNF-?protein content and IL-1?and TNF-?mRNA in the serum of Lactacystin induced neuron-like PC12 cellsThe results show that:The contents of IL-1?and TNF-?proteins in the supernatant solution of cells in the sericyb group were studied and The expression of IL-1?and TNF-?mediated mRNA in cells was lower than that in the model group(p<0.01).The content of IL-1?complement and TNF-?protein in the supernatant solution of cells and The expressions of IL-1?and TNF-?mRNA in cells were higher than those in the alone coumarin group(p<0.01).4.3 the effect of the coumarin on the expression of IL-1?,TNF-?protein and IL-1?and TNF-?mRNA in the serum of Lactacystin induced neuron-like PC12 cellsThe results show that:Compared with the model group,The contents of IL-1?purge and TNF-?protein in the supernatant solution of cells and the expressions of IL-1?purge and TNF-?mRNA in cells all decreased significantly(p<0.01).Compared with the sericyb group,the contents of IL-1?purge and TNF-?proteins and the expression of IL-1?purge and TNF-?pruning mRNA in cells were all lower than that in the sericyb group(p<0.01).Conclusion:1.In this experiment,Lactacystin proteinase inhibitor was used to successfully simulate the model of neuronal cell damage in Parkinson's disease.In the model group,the activity of PC12 cells decreased,and the expression of inflammatory cytokines IL-1?,TNF-?protein and IL-1?and TNF-?mRNA were all increased.2.The single active coumarin containing drug serum can promote the proliferation of Lactacystin induced neuron-like PC12 cells;In addition,the serum of the coumarin can lower the expression of the inflammatory cytokines IL-1?,TNF-?protein,and the expression of IL-1?and TNF-?mRNA in Lactacystin induced neuronal PC12 cells.3.Coumarin drug-containing serum on Lactacystin induced neuronal PC12 cell plays a protective role,with its downgrade IL-1?cell inflammation factor,the content of TNF-?protein,TNF-?and IL-1?mRNA expression,inhibit the neuronal PC12 cell induced by Lactacystin inflammatory response are closely related.